Methods and means for treating dna repeat instability associated genetic disorders

ABSTRACT

The current invention provides for methods and medicaments that apply oligonucleotide molecules complementary only to a repetitive sequence in a human gene transcript, for the manufacture of a medicament for the diagnosis, treatment or prevention of a cis-element repeat instability associated genetic disorders in humans. The invention hence provides a method of treatment for cis-element repeat instability associated genetic disorders. The invention also pertains to modified oligonucleotides which can be applied in method of the invention to prevent the accumulation and/or translation of repeat expanded transcripts in cells.

FIELD OF THE INVENTION

The current invention relates to the field of medicine, in particular tothe treatment of genetic disorders associated with genes that haveunstable repeats in their coding or non-coding sequences, most inparticular unstable repeats in the human Huntington disease causing HDgene or the myotonic dystrophy type 1 causing DMPK gene.

BACKGROUND OF THE INVENTION

Instability of gene-specific microsatellite and minisatellite repetitivesequences, leading to increase in length of the repetitive sequences inthe satellite, is associated with about 35 human genetic disorders.Instability of trinucleotide repeats is for instance found in genescausing X-linked spinal and bulbar muscular atrophy (SBMA), myotonicdystrophy type 1 (DM1), fragile X syndrome (FRAX genes A, E, F),Huntington's disease (HD) and several spinocerebellar ataxias (SCA genefamily). Unstable repeats are found in coding regions of genes, such asthe Huntington's disease gene, whereby the phenotype of the disorder isbrought about by alteration of protein function and/or protein folding.Unstable repeat units are also found in untranslated regions, such as inmyotonic dystrophy type 1 (DM1) in the 3′ UTR or in intronic sequencessuch as in myotonic dystrophy type 2 (DM2). The normal number of repeatsis around 5 to 37 for DMPK, but increases to premutation and fulldisease state two to ten fold or more, to 50, 100 and sometimes 1000 ormore repeat units. For DM2/ZNF9 increases to 10,000 or more repeats havebeen reported. (Cleary and Pearson, Cytogenet. Genome Res. 100: 25-55,2003).

The causative gene for Huntington's disease, HD, is located onchromosome 4. Huntington's disease is inherited in an autosomal dominantfashion. When the gene has more than 35 CAG trinucleotide repeats codingfor a polyglutamine stretch, the number of repeats can expand insuccessive generations. Because of the progressive increase in length ofthe repeats, the disease tends to increase in severity and presents atan earlier age in successive generations, a process called anticipation.The product of the, HD gene is the 348 kDa cytoplasmic proteinhuntingtin. Huntingtin has a characteristic sequence of fewer than 40glutamine amino acid residues in the normal form; the mutated huntingtincausing the disease has more than 40 residues. The continuous expressionof mutant huntingtin molecules in neuronal cells results in theformation of large protein deposits which eventually give rise to celldeath, especially in the frontal lobes and the basal ganglia (mainly inthe caudate nucleus). The severity of the disease is generallyproportional to the number of extra residues.

DM1 is the most common muscular dystrophy in adults and is an inherited,progressive, degenerative, multisystemic disorder of predominantlyskeletal muscle, heart and brain. DM1 is caused by expansion of anunstable trinucleotide (CTG)n repeat in the 3′ untranslated region ofthe DMPK gene (myotonic dystrophy protein kinase) on human chromosome19q (Brook et al, Cell, 1992). Type 2 myotonic dystrophy (DM2) is causedby a CCTG expansion in intron 1 of the ZNF9 gene, (Liguori et al,Science 2001). In the case of myotonic dystrophy type 1, thenuclear-cytoplasmic export of DMPK transcripts is blocked by theincreased length of the repeats, which form hairpin-like secondarystructures that accumulate in nuclear foci. DMPK transcripts bearing along (CUG)n tract can form hairpin-like structures that bind proteins ofthe muscleblind family and subsequently aggregate in ribonuclear foci inthe nucleus. These nuclear inclusions are thought to sequestermuscleblind proteins, and potentially other factors, which then becomelimiting to the cell. In DM2, accumulation of ZNF9 RNA carrying the(CCUG)n expanded repeat form similar foci. Since muscleblind proteinsare splicing factors, their depletion results in a dramaticrearrangement in splicing of other transcripts. Transcripts of manygenes consequently become aberrantly spliced, for instance by inclusionof fetal exons, or exclusion of exons, resulting in non-functionalproteins and impaired cell function.

The observations and new insights above have led to the understandingthat unstable repeat diseases, such as myotonic dystrophy type 1,Huntington's disease and others can be treated by removing, either fullyor at least in part, the aberrant transcript that causes the disease.For DM1, the aberrant transcript that accumulates in the nucleus couldbe down regulated or fully removed. Even relatively small reductions ofthe aberrant transcript could release substantial and possiblysufficient amounts of sequestered cellular factors and thereby help torestore normal RNA processing and cellular metabolism for DM (Kanadia etal., PNAS 2006). In the case of HD, a reduction in the accumulation ofhuntingtin protein deposits in the cells of an HD patient can amelioratethe symptoms of the disease.

A few attempts have been made to design methods of treatment andmedicaments for unstable repeat disease myotonic dystrophy type 1 usingantisense nucleic acids, RNA interference or ribozymes. (i) Langlois etal. (Molecular Therapy, Vol. 7 No. 5, 2003) designed a ribozyme capableof cleaving DMPK mRNA. The hammerhead ribozyme is provided with astretch RNA complementary to the 3′ UTR of DMPK just before the CUGrepeat. In vivo, vector transcribed ribozyme was capable of cleaving anddiminishing in transfected cells both the expanded CUG repeat containingmRNA as well as the normal mRNA species with 63 and 50% respectively.Hence, also the normal transcript is gravely affected by this approachand the affected mRNA species with expanded repeats are not specificallytargeted.

(ii) Another approach was taken by Langlois et al., (Journal BiologicalChemistry, vol 280, no. 17, 2005) using RNA interference. Alentivirus-delivered short-hairpin RNA (shRNA) was introduced in DM1myoblasts and demonstrated to down regulate nuclear retained mutant DMPKmRNAs. Four shRNA molecules were tested, two were complementary againstcoding regions of DMPK, one against a unique sequence in the 3′ UTR andone negative control with an irrelevant sequence. The first two shRNAswere capable of down regulating the mutant DMPK transcript with theamplified repeat to about 50%, but even more effective in downregulating the cytoplasmic wildtype transcript to about 30% or less.Equivalent synthetic siRNA delivered by cationic lipids was ineffective.The shRNA directed at the 3′ UTR sequence proved to be ineffective forboth transcripts. Hence, also this approach is not targeted selectivelyto the expanded repeat mRNA species.

(iii) A third approach by Furling et al. (Gene Therapy, Vol. 10, p795-802, 2003) used a recombinant retrovirus expressing a 149-bp longantisense RNA to inhibit DMPK mRNA levels in human DM1 myoblasts. Aretrovirus was designed to provide DM1 cells with the 149 by longantisense RNA complementary to a 39 bp-long (CUG)13 repeat and a 110 byregion following the repeat to increase specificity. This method yieldeda decrease in mutated (repeat expanded) DMPK transcript of 80%, comparedto a 50% reduction in the wild type DMPK transcript and restoration ofdifferentiation and functional characteristics in infected DM1myoblasts. Hence, also this approach is not targeted selectively to theexpanded repeat mRNA species, it depends on a very long antisense RNAand can only be used in combination with recombinant viral deliverytechniques.

DETAILED DESCRIPTION OF THE INVENTION

The methods and techniques described above provide nucleid acid basedmethods that cause non-selective breakdown of both the affected repeatexpanded allele and unaffected (normal) allele for genetic diseases thatare associated with repeat instability and/or expansion. Moreover, theart employs sequences specific for the gene associated with the diseaseand does not provide a method that is applicable to several geneticdisorders associated with repeat expansion. Finally, the art onlyteaches methods that involve use of recombinant DNA vector deliverysystems, which need to be adapted for each oligonucleotide and targetcell and which still need to be further optimised.

The current invention provides a solution for these problems by using ashort single stranded nucleic acid molecule that comprises or consistsof a sequence, which is complementary to the expanded repeat regiononly, i.e. it does not rely on hybridisation to unique sequences inexons or introns of the repeat containing gene. Furthermore, it is notessential that the employed nucleic acid (oligonucleotide) reducestranscripts by the RNAse H mediated breakdown mechanism.

Without wishing to be bound by theory, the current invention may cause adecrease in transcript levels by alterations in posttranscriptionalprocessing and/or splicing of the premature RNA. A decrease intranscript levels via alternative splicing and/or postranscriptionalprocessing is thought to result in transcripts lacking the overlyexpanded or instable (tri)nucleotide repeat, but still possessingfunctional activities. The reduction of aberrant transcripts by alteredRNA processing and/or splicing may prevent accumulation and/ortranslation of aberrant, repeat expanded transcripts in cells.

Without wishing to be bound by theory the method of the currentinvention is also thought to provide specificity for the affectedtranscript with the expanded repeat because the kinetics forhybridisation to the expanded repeat are more favourable. The likelihoodthat a repeat specific complementary nucleic acid oligonucleotidemolecule will hybridise to a complementary stretch in an RNA or DNAmolecule increases with the size of the repetitive stretch. RNAmolecules and in particular RNA molecules comprising repetitivesequences are normally internally paired, forming a secondary structurecomprising open loops and closed hairpin parts. Only the open parts arerelatively accessible for complementary nucleic acids. The short repeatstretches of a wild type transcript not associated with disease is oftenonly 5 to about 20-40 repeats and due to the secondary structurerelatively inaccessible for base pairing with a complementary nucleicacid. In contrast, the repeat units of the expanded repeat and diseaseassociated allele is normally at least 2 fold expanded but usually evenmore, 3, 5, 10 fold, up to 100 or even more than 1000 fold expansion forsome unstable repeat disorders. This expansion increases the likelihoodthat part of the repeat is, at least temporarily, in an open loopstructure and thereby more accessible to base pairing with acomplementary nucleic acid molecule, relative to the wild type allele.So despite the fact that the oligonucleotide is complementary to arepeat sequence present in both wildtype and repeat-expanded transcriptsand could theoretically hybridise to both transcripts, the currentinvention teaches that oligonucleotides complementary to the repetitivetracts preferably hybridise to the disease-associated or disease-causingtranscripts and leave the function of normal transcripts relativelyunaffected. This selectivity is beneficial for treating diseasesassociated with repeat instability irrespective of the mechanism ofreduction of the aberrant transcript.

The invention thus provides a method for the treatment of unstablecis-element DNA repeat associated genetic disorders, by providingnucleic acid molecules that are complementary to and/or capable ofhybridising to the repetitive sequences only. This method therebypreferentially targets the expanded repeat transcripts and leaves thetranscripts of the normal, wild type allele relatively unaffected. Thisis advantageous since the normal allele can thereby provide for thenormal function of the gene, which is at least desirable and, dependingon the particular gene with unstable DNA repeats, may in many cases beessential for the cell and/or individual to be treated.

Furthermore, this approach is not limited to a particular unstable DNArepeat associated genetic disorder, but may be applied to any of theknown unstable DNA repeat diseases, such as, but not limited to: codingregions repeat diseases having a polyglutamine (CAG) repeat:Huntington's disease, Haw River syndrome, Kennedy's disease/spinobulbarmuscular atrophy, spino-cerebellar ataxia, or diseases havingpolyalanine (GCG) repeats such as: infantile spasm syndrome,deidocranial dysplasia, blepharophimosis/ptosis/epicanthus invensussyndrome, hand-foot-genital syndrome, synpolydactyly, oculopharyngealmuscular dystrophy, holoprosencephaly. Diseases with repeats innon-coding regions of genes to be treated according to the inventioncomprise the trinucleotide repeat disorders (mostly CTG and/or CAGand/or CCTG repeats): myotonic dystrophy type 1, myotonic dystrophy type2. Friedreich's ataxia (mainly GAA), spino-cerebellar ataxia, autism.Furthermore, the method of the invention can be applied to fragile siteassociated repeat disorder comprising various fragile X-syndromes,Jacobsen syndrome and other unstable repetitive element disorders suchas myoclonus epilepsy, facioscapulohumeral dystrophy and certain formsof diabetes mellitus type 2.

Another advantage of the current invention is that the oligonucleotidesspecific for a repeat region may be administered directly to cells andit does not rely on vector-based delivery systems. The techniquesdescribed in the prior art, for instance those mentioned above fortreatment of DM1 and removal of DMPK transcripts from cells, require theuse of vector based delivery systems to administer sufficient levels ofoligonucleotides to the cell. The use of plasmid or viral vectors is yetless desirable for therapeutic purposes because of current strict safetyregulations for therapeutic recombinant DNA vectors, the production ofsufficient recombinant vectors for broad clinical application and thelimited control and reversibility of an exaggerated (or non-specific)response after application. Nevertheless, optimisation in future islikely in these areas and viral delivery of plasmids could yield anadvantageous long lasting effect. The current inventors havesurprisingly found that oligonucleotides that comprise or consist of asequence that is complementary to repetitive sequences of expandedrepeat transcripts, due to the expansion of their molecular target forhybridisation, have a much increased affinity and/or avidity for theirtarget in comparison to oligonucleotides that are specific for uniquesequences in a transcript. Because of this high affinity and avidity forthe repeat expanded target transcript, lower amounts of oligonucleotidesuffice to yield sufficient inhibition and/or reduction of the repeatexpanded allele by RNase H degradation, RNA interference degradation oraltered post-transcriptional processing (comprising but not limited tosplicing or exon skipping) activities. The oligonucleotides of thecurrent invention which are complementary to repetitive sequences only,may be produced synthetically and are potent enough to be effective whendelivered directly to cells using commonly applied techniques for directdelivery of oligonucleotides to cells and/or tissues. Recombinant vectordelivery systems may, when desired, be circumvented by using the methodand the oligonucleotide molecules of the current invention.

In a first aspect, the current invention discloses and teaches the useof an oligonucleotide comprising or consisting of a sequence that iscomplementary only to a repetitive sequence in a human gene transcriptfor the manufacture of a medicament for the diagnosis, treatment orprevention of a cis-element repeat instability associated geneticdisorders in humans. The invention hence provides a method of treatmentfor cis-element repeat instability associated genetic disorders.

In a second aspect, the invention also pertains to an oligonucleotidewhich can be used in the first aspect of the invention and/or applied inmethod of the invention to prevent the accumulation and/or translationof repeat expanded transcripts in cells.

An oligonucleotide of the invention may comprise a sequence that iscomplementary only to a repetitive sequence as defined below.Preferably, the repetitive sequence is at least 50% of the length of theoligonucleotide of the invention, more preferably at least 60%, evenmore preferably at least 70%, even more preferably at least 80%, evenmore preferably at least 90% or more. In a most preferred embodiment,the oligonucleotide of the invention consists of a sequence that iscomplementary only to a repetitive sequence as defined below. Forexample, an oligonucleotide may comprise a sequence that iscomplementary only to a repetitive sequence as defined below and atargeting part, which is later on called a targeting ligand.

A repeat or repetitive element or repetitive sequence or repetitivestretch is herein defined as a repetition of at least 3, 4, 5, 10, 100,1000 or more, of a repetitive unit or repetitive nucleotide unit orrepeat nucleotide unit comprising a trinucleotide repetitive unit, oralternatively a 4, 5 or 6 nucleotide repetitive unit, in a transcribedgene sequence in the genome of a subject, including a human subject.

An oligonucleotide may be single stranded or double stranded. Doublestranded means that the oligonucleotide is an heterodimer made of twocomplementary strands, such as in a siRNA. In a preferred embodiment, anoligonucleotide is single stranded. A single stranded oligonucleotidehas several advantages compared to a double stranded siRNAoligonucleotide: (i) its synthesis is expected to be easier than twocomplementary siRNA strands; (ii) there is a wider range of chemicalmodifications possible to optimise more effective uptake in cells, abetter (physiological) stability and to decrease potential genericadverse effects; and (iii) siRNAs have a higher potential fornon-specific effects and exaggerated pharmacology (e.g. less controlpossible of effectiveness and selectivity by treatment schedule or dose)and (iv) siRNAs are less likely to act in the nucleus and cannot bedirected against introns. Therefore, in a preferred embodiment of thefirst aspect, the invention relates to the use of a single strandedoligonucleotide comprising or consisting of a sequence that iscomplementary only to a repetitive sequence in a human gene transcriptfor the manufacture of a medicament for the diagnosis, treatment orprevention of a cis-element repeat instability associated geneticdisorders in humans.

The oligonucleotide(s) preferably comprise at least 10 to about 50consecutive nucleotides complementary to a repetitive element, morepreferably 12 to 45 nucleotides, even more preferably 12 to 30, and mostpreferably 12 to 25 nucleotides complementary to a repetitive stretch,preferably having a trinucleotide repeat unit or a tetranucleotiderepeat unit. The oligonucleotide may be complementary to and/or capableof hybridizing to a repetitive stretch in a coding region of atranscript, preferably a polyglutamine (CAG) or a polyalanine (GCG)coding tract. The oligonucleotide may also be complementary to and/orcapable of hybridizing to a non-coding region for instance 5′ or 3′untranslated regions, or intronic sequences present in precursor RNAmolecules.

In a preferred embodiment the oligonucleotide to be used in the methodof the invention comprises or consists of a sequence that iscomplementary to a repetitive element having as repetitive nucleotideunit a repetitive nucleotide unit selected from the group consisting of(CAG)n, (GCG)n, (CUG)n, (CGG)n (GAA)n, (GCC)n and (CCUG)n. and saidoligonucleotide being a single or double stranded oligonucleotide.Preferably, the oligonucleotide is double stranded.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a polyglutamine (CAG)n tract in a transcript isparticularly useful for the diagnosis, treatment and/or prevention ofthe human disorders Huntington's disease, several forms ofspino-cerebellar ataxia or Haw River syndrome, X-linked spinal andbulbar muscular atrophy and/or dentatorubral-pallidoluysian atrophycaused by repeat expansions in the HD, HDL2/JPH3, SBMA/AR, SCA1/ATX1,SCA2/ATX2, SCA3/ATX3, SCA6/CACNAIA, SCAT, SCA17, AR or DRPLA humangenes.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a polyalanine (GCG)n tract in a transcript isparticularly useful for the diagnosis, treatment and/or prevention ofthe human disorders: infantile spasm syndrome, deidocranial dysplasia,blepharophimosis, hand-foot-genital disease, synpolydactyl),oculopharyngeal muscular dystrophy and/or holoprosencephaly, which arecaused by repeat expansions in the ARX, CBFA1, FOXL2, HOXA13, HOXD13,OPDM/PABP2, TCFBR1 or ZIC2 human genes.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a (CUG)n repeat in a transcript and isparticularly useful for the diagnosis, treatment and/or prevention ofthe human genetic disorder myotonic dystrophy type 1, spino-cerebrellarataxia 8 and/or Huntington's disease-like 2 caused by repeat expansionsin the DM1/DMPK, SCA8 or JPH3 genes respectively. Preferably, thesegenes are from human origin.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a (CCUG)n repeat in a transcript isparticularly useful for the diagnosis, treatment and/or prevention ofthe human genetic disorder myotonic dystrophy type 2, caused by repeatexpansions in the DM2/ZNF9 gene.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a (CGG)n repeat in a transcript is particularlyuseful for the diagnosis, treatment and/or prevention of human fragile Xsyndromes, caused by repeat expansion in the FRAXA/FMR1, FRAXE/FMR2 andFRAXF/FAM11A genes.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a (CCG)n repeat in a transcript is particularlyuseful for the diagnosis, treatment and/or prevention of the humangenetic disorder Jacobsen syndrome, caused by repeat expansion in theFRA11B/CBL2 gene.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a (GAA)n repeat in a transcript is particularlyuseful for the diagnosis, treatment and/or prevention of the humangenetic disorder Friedreich's ataxia.

The use of an oligonucleotide that comprises or consists of a sequencethat is complementary to a (ATTCT)n repeat in an intron is particularlyuseful for the diagnosis, treatment and/or prevention of the humangenetic disorder Spinocerebellar ataxia type 10 (SCA10).

The repeat-complementary oligonucleotide to be used in the method of theinvention may comprise or consist of RNA, DNA, Locked nucleic acid(LNA), peptide nucleic acid (PNA), morpholino phosphorodiamidates (PMO),ethylene bridged nucleic acid (ENA) or mixtures/hybrids thereof thatcomprise combinations of naturally occurring DNA and RNA nucleotides andsynthetic, modified nucleotides. In such oligonucleotides, thephosphodiester backbone chemistry may further be replaced by othermodifications, such as phosphorothioates or methylphosphonates. Otheroligonucleotide modifications exist and new ones are likely to bedeveloped and used in practice. However, all such oligonucleotides havethe character of an oligomer with the ability of sequence specificbinding to RNA. Therefore in a preferred embodiment, the oligonucleotidecomprises or consists of RNA nucleotides, DNA nucleotides, lockednucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides,morpholino phosphorodiamidates, ethylene-bridged nucleic acid (ENA)nucleotides or mixtures thereof with or without phosphorothioatecontaining backbones.

Oligonucleotides containing at least in part naturally occurring DNAnucleotides are useful for inducing degradation of DNA-RNA hybridmolecules in the cell by RNase H activity (EC.3.1.26.4).

Naturally occurring RNA ribonucleotides or RNA-like syntheticribonucleotides comprising oligonucleotides may be applied in the methodof the invention to form double stranded RNA-RNA hybrids that act asenzyme-dependent antisense through the RNA interference or silencing(RNAi/siRNA) pathways, involving target RNA recognition throughsense-antisense strand pairing followed by target RNA degradation by theRNA-induced silencing complex (RISC).

Alternatively or in addition, steric blocking antisense oligonucleotides(RNase-H independent antisense) interfere with gene expression or otherprecursor RNA or messenger RNA-dependent cellular processes, inparticular but not limited to RNA splicing and exon skipping, by bindingto a target sequence of RNA transcript and getting in the way ofprocesses such as translation or blocking of splice donor or spliceacceptor sites. Alteration of splicing and exon skipping techniquesusing modified antisense oligonucleotides are well documented, known tothe skilled artisan and may for instance be found in U.S. Pat. No.6,210,892, WO9426887, WO04/083446 and WO02/24906. Moreover, sterichindrance may inhibit the binding of proteins, nuclear factors andothers and thereby contribute to the decrease in nuclear accumulation orribonuclear foci in diseases like DM1.

The oligonucleotides of the invention, which may comprise synthetic ormodified nucleotides, complementary to (expanded) repetitive sequencesare useful for the method of the invention for reducing or inactivatingrepeat containing transcripts via the siRNA/RNA interference orsilencing pathway.

Single or double stranded oligonucleotides to be used in the method ofthe invention may comprise or consist of DNA nucleotides, RNAnucleotides, 2′-O substituted ribonucleotides, including alkyl andmethoxy ethyl substitutions, peptide nucleic acid (PNA), locked nucleicacid (LNA) and morpholino (PMO) antisense oligonucleotides andethylene-bridged nucleotides (ENA) and combinations thereof, optionallychimeras with RNAse H dependent antisense. Integration of locked nucleicacids in the oligonucleotide changes the conformation of the helix afterbase pairing and increases the stability of the duplex. Integration ofLNA bases into the oligonucleotide sequence can therefore be used toincrease the ability of complementary oligonucleotides of the inventionto be active in vitro and in vivo to increase RNA degradation inhibitaccumulation of transcripts or increase exon skipping capabilities.Peptide nucleic acids (PNAs), an artificial DNA/RNA analog, in which thebackbone is a pseudopeptide rather than a sugar, have the ability toform extremely stable complexes with complementary DNA oligomers, byincreased binding and a higher melting temperature. Also PNAs aresuperior reagents in antisense and exon skipping applications of theinvention. Most preferably, the oligonucleotides to be used in themethod of this invention comprise, at least in part or fully,2′-O-methoxy ethyl phosphorothioate RNA nucleotides or 2′-O-methylphosphorothioate RNA nucleotides. Oligonucleotides comprising orconsisting of a sequence that is complementary to a repetitive sequenceselected from the group consisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n,(CCG)n, (GAA)n, (GCC)n and (CCUG)n having a length of 10 to 50, morepreferably 12 to 35, most preferably 12 to 25 nucleotides, andcomprising 2′-β-methoxyethyl phosphorothioate RNA nucleotides,2′-O-methyl phosphorothioate RNA nucleotides, LNA nucleotides or PMOnucleotides are most preferred for use in the invention for thediagnosis, treatment of prevention of cis-element repeat instabilitygenetic disorders.

Accordingly, in a preferred embodiment, an oligonucleotide of theinvention and used in the invention comprises or consists of a sequencethat is complementary to a repetitive sequence selected from the groupconsisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and(CCUG)n, has a length of 10 to 50 nucleotides and is furthercharacterized by:

-   -   a) comprising 2′-O-substituted RNA phosphorothioate nucleotides        such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothiote        nucleotides, LNA nucleotides or PMO nucleotides. The nucleotides        could be used in any combination and/or with DNA        phosphorothioate or RNA nucleotides; and/or    -   b) being a single stranded oligonucleotide.

Accordingly, in another preferred embodiment, an oligonucleotide of theinvention and used in the invention comprises or consists of a sequencethat is complementary to a repetitive sequence selected from the groupconsisting of (CAG)n, (GCG)n, (CUG)n, (CGG)n, (GAA)n, (GCC)n and(CCUG)n, has a length of 10 to 50 nucleotides and is furthercharacterized by:

-   -   c) comprising 2′-O-substituted RNA phosphorothioate nucleotides        such as 2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothiote        nucleotides, LNA nucleotides or PMO nucleotides. The nucleotides        could be used in combination and/or with DNA phosphorothioate or        RNA nucleotides; and/or    -   d) being a double stranded oligonucleotide.

In case, the invention relates to a double stranded oligonucleotide withtwo complementary strands, the antisense strand, complementary only to arepetitive sequence in a human gene transcript, this double strandedoligonucleotide is preferably not the siRNA with antisense RNA strand(CUG)₇ and sense RNA strand (GCA)₇ applied to cultured monkey fibroblast(COS-7) or human neuroblastoma (SH-SY5Y) cell lines with or withouttransfection with a human Huntington gene exon 1 fused to GFP and asdepicted in Wanzhao Liu et al (Wanzhao Liu et al, (2003), Proc. JapanAcad, 79: 293-298). More preferably, the invention does neither relateto the double stranded oligonucleotide siRNA (with antisense strand(CUG)₇ and sense strand (GCA)₇) nor to its use for the manufacture of amedicament for the treatment or prevention of Huntington disease, evenmore preferably for the treatment or prevention of Huntington diseasegene exon 1 containing construct.

Although use of a single oligonucleotide may be sufficient for reducingthe amount of repeat expanded transcripts, such as nuclear accumulatedDMPK or ZNF9 transcripts or segments thereof or sufficient reduction ofaccumulation of repeat expanded HD protein, it is also within the scopeof the invention to combine 2, 3, 4, 5 or more oligonucleotides. Theoligonucleotide comprising or consisting of a sequence that iscomplementary to a repetitive part of a transcript may be advantageouslycombined with oligonucleotides that comprise or consist of sequencesthat are complementary to and/or capable of hybridizing with uniquesequences in a repeat containing transcript. The method of the inventionand the medicaments of the invention comprising repeat specificoligonucleotides may also be combined with any other treatment ormedicament for cis-element repeat instability genetic disorders. Fordiagnostic purposes the oligonucleotide used in the method of theinvention may be provided with a radioactive label or fluorescent labelallowing detection of transcripts in samples, in cells in situ in vivo,ex vivo or in vitro. For myotonic dystrophy, labelled oligonucleotidesmay be used for diagnostic purposes, for visualisation of nuclearaggregates of DMPK or ZNF9 RNA transcript molecules with associatedproteins. Fluorescent labels may comprise Cy3, Cy5, FITC, TRITC,Rhodamine, GFP and the like. Radioactive labels may comprise ³H, ³⁵S,^(32/33)P, ¹²⁵I. Enzymes and/or immunogenic haptens such as digoxigenin,biotin and other molecular tags (HA, Myc, FLAG, VSV, lexA) may also beused. Accordingly, in a further aspect, the invention discloses an vitroor ex vivo detection and/or diagnostic method wherein a oligonucleotideas defined above is used.

The oligonucleotides for use according to the invention are suitable fordirect administration to cells, tissues and/or organs in vivo ofindividuals affected by or at risk of developing a cis-element repeatinstability disorder, and may be administered directly in vivo, ex vivoor in vitro. Alternatively, the oligonucleotides may be provided by anucleic acid vector capable of conferring expression of theoligonucleotide in human cells, in order to allow a sustainable sourceof the oligonucleotides. Oligonucleotide molecules according to theinvention may be provided to a cell, tissue, organ and/or subject to betreated in the form of an expression vector that is capable ofconferring expression of the oligonucleotide in human cells. The vectoris preferably introduced in the cell by a gene delivery vehicle.Preferred vehicles for delivery are viral vectors such as retroviralvectors, adeno-associated virus vectors (AAV), adenoviral vectors,Semliki Forest virus vectors (SFV), EBV vectors and the like. Alsoplasmids, artificial chromosomes, plasmids suitable for targetedhomologous recombination and integration in the human genome of cellsmay be suitably applied for delivery of oligonucleotides. Preferred forthe current invention are those vectors wherein transcription is drivenfrom polIII promoters, and/or wherein transcripts are in the formfusions with U1 or U7 transcripts, which yield good results fordelivering small transcripts.

In a preferred embodiment, a concentration of oligonucleotide, which isranged between about 0.1 nM and about 1 μM is used. More preferably, theconcentration used is ranged between about 0.3 to about 400 nM, evenmore preferably between about 1 to about 200 nM. If severaloligonucleotides are used, this concentration may refer to the totalconcentration of oligonucleotides or the concentration of eacholigonucleotide added. The ranges of concentration of oligonucleotide(s)as given above are preferred concentrations for in vitro or ex vivouses. The skilled person will understand that depending on theoligonueleotide(s) used, the target cell to be treated, the gene targetand its expression levels, the medium used and the transfection andincubation conditions, the concentration of oligonucleotide(s) used mayfurther vary and may need to be optimised any further.

More preferably, the oligonucleotides to be used in the invention toprevent, treat or diagnose cis-element repeat instability disorders aresynthetically produced and administered directly to cells, tissues,organs and/or patients in formulated form in pharmaceutically acceptablecompositions. The delivery of the pharmaceutical compositions to thesubject is preferably carried out by one or more parenteral injections,e.g. intravenous and/or subcutaneous and/or intramuscular and/orintrathecal and/or intraventricular administrations, preferablyinjections, at one or at multiple sites in the human body. Anintrathecal or intraventricular administration (in the cerebrospinalfluid) is preferably realized by introducing a diffusion pump into thebody of a subject. Several diffusion pumps are known to the skilledperson.

Pharmaceutical compositions that are to be used to target theoligonucleotide molecules comprising or consisting of a sequence that iscomplementary to repetitive sequences may comprise various excipientssuch as diluents, fillers, preservatives, solubilisers and the like,which may for instance be found in Remington: The Science and Practiceof Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams &Wilkins, 2000.

Particularly preferred for the method of the invention is the use ofexcipients that will aid in delivery of the oligonucleotides to thecells and into the cells, in particular excipients capable of formingcomplexes, vesicles and/or liposomes that deliver substances and/oroligonucleotide(s) complexed or trapped in the vesicles or liposomesthrough a cell membrane. Many of these substances are known in the art.Suitable substances comprise polyethylenimine (PEI), ExGen 500,synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsidproteins that are capable of self assembly into particles that candeliver oligonucleotides to cells. Lipofectin represents an example ofliposomal transfection agents. It consists of two lipid components, acationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and aneutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutralcomponent mediates the intracellular release. Another group of deliverysystems are polymeric nanoparticles. Polycations such likediethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNAtransfection reagent can be combined with butylcyanoacrylate (PBCA) andhexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that candeliver oligonucleotides across cell membranes into cells. In additionto these common nanoparticle materials, the cationic peptide protamineoffers an alternative approach to formulate oligonucleotides ascolloids. This colloidal nanoparticle system can form so calledproticles, which can be prepared by a simple self-assembly process topackage and mediate intracellular release of the oligonucleotides. Theskilled person may select and adapt any of the above or othercommercially available alternative excipients and delivery systems topackage and deliver oligonucleotides for use in the current invention todeliver oligonucleotides for the treatment of cis-element repeatinstability disorders in humans.

In addition, the oligonucleotide could be covalcntly or non-covalcntlylinked to a targeting ligand specifically designed to facilitate theuptake in to the cell, cytoplasm and/or its nucleus. Such ligand couldcomprise (i) a compound (including but not limited to peptide(-like)structures) recognising cell, tissue or organ specific elementsfacilitating cellular uptake and/or (ii) a chemical compound able tofacilitate the uptake in to cells and/or the intracellular release of anoligonucleotide from vesicles, e.g. endosomes or lysosomes. Suchtargeting ligand would also encompass molecules facilitating the uptakeof oligonucleotides into the brain through the blood brain barrier.Therefore, in a preferred embodiment, an oligonucleotide in a medicamentis provided with at least an excipient and/or a targeting ligand fordelivery and/or a delivery device of the oligonucleotide to cells and/orenhancing its intracellular delivery. Accordingly, the invention alsoencompasses a pharmaceutically acceptable composition comprising anoligonucleotide of the invention and further comprising at least oneexcipient and/or a targeting ligand for delivery and/or a deliverydevice of the oligonucleotide to the cell and/or enhancing itsintracellular delivery.

The invention also pertains to a method for the reduction of repeatcontaining gene transcripts in a cell comprising the administration of asingle strand or double stranded oligonucleotide molecule, preferablycomprising 2′-O-substituted RNA phosphorothioate nucleotides such as2′-O-methyl or 2′-O-methoxy ethyl RNA phosphorothioate nucleotides orLNA nucleotides or PMO nucleotides, and having a length of 10 to 50nucleotides that are complementary to the repetitive sequence only. Thenucleotides could be used in combination and/or with DNAphosphorothioate nucleotides.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but combinations and/or items notspecifically mentioned are not excluded. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the element is present, unless thecontext clearly requires that there be one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one”.

FIGURE LEGENDS

FIG. 1: Northern blot of RNA isolated from myotubes transfected withdifferent oligonucleotides or mock control. The myotubes were derivedfrom immorto mouse myoblast cell lines containing a transgenic humanDMPK genes with (CTG)n repeat expansion length of approximately 500 nextto its normal mouse DMPK gene without (CTG) repeat. The blot shows humanDMPK mRNA (top), mouse DMPK (mDMPK) mRNA (middle) and mouse GAPDH mRNA(bottom).

FIG. 2: The human and mouse DMPK signals of FIG. 1 were quantified byphosphoimager analysis and normalized, to the GAPDH signal. The resultsare expressed relative to the mock treatment (set to 100).

FIG. 3: Northern blot of total RNA isolated from murine myotubescontaining a mouse-human chimaeric DMPK gene in which the 3′ part of themDMPK gene was replaced by the cognate segment of the human DMPK geneincluding a (CTG)₁₁₀-repeat. The blot was probed for DMPK mRNA (upperpanel) and mouse GAPDH mRNA (bottom). Cells were transfected withantisense oligonucleotide PS58 or control.

FIG. 4 shows the response of DM500 myotubes treated with variousconcentrations of oligonucleotide PS58. The expression of hDMPK wasquantified via Northern blot analysis followed by phosphoimageranalysis. The signal was normalised to the GAPDH signal and expressedrelative to the response after mock treatment.

FIG. 5 shows the Northern blot of total RNA of DM500 myotubestransfected with 200 nM PS58 at different time points: 2 h, 4 h, 8 h and48 h before harvesting. Mock treatment was performed 48 h beforeharvesting. Northern blots show human and mouse DMPK and mouse GAPDHmRNA. These were quantified by phosphoimager and the normalized DMPKsignal was expressed relative to mock treatment.

FIG. 6 shows the Northern blot of total RNA of DM500 myotubes harvested2 d, 4 d, 6 d and 8 d after transfection with 200 nM PS58 or mockcontrol. Northern blot analysis and quantification was performed asbefore.

FIG. 7 shows a Northern blot of total RNA from MyoD-transformedmyoblasts treated with oligonucleotide PS58 (20 and 200 nM) or mockcontrol. The myoblasts were derived from fibroblasts obtained from acongenital myotonic dystrophy type I patient bearing a hDMPK allele witha triplet repeat expansion length of approximately 1500 and a hDMPKallele with normal length of 11 repeats. The Northern blot washybridized with a human DMPK (hDMPK) probe and GAPDH mRNA probe. Thehuman DMPK signals were normalized to the GAPDH signal and expressedrelative to mock control.

FIG. 8 shows the RT-PCR analysis of DM500 myotubes transfected with20011M of oligonucleotide PS58, specific to the (CUG) repeat sequenceonly, oligonucleotide PS113, specific to a sequence in exon 1, or mockcontrol. RT-PCR analysis was performed with primers specific for hDMPKmRNA and three other gene transcripts with a naturally occurring (CUG)repeat in mice: Ptbp1 mRNA with a (CUG)6, Syndecan3 mRNA with a (CUG)6and Taxilinbeta mRNA with a (CUG)9. The intensity of the signals werenormalized to the actin signal and expressed relative to mock control.

FIG. 9 shows FISH analysis of DM500 myoblasts transfected with 200 nMPS58 (B) or mock control (A). Fourty eight hours after the start of thetreatment, the cells were washed and fixed and subsequently hybridizedwith fluorescently labeled oligonucleotide Cy3-(CAG)10-Cy3. Theribonuclear foci indicative of hDMPK (CUG)₅₀₀ mRNA aggregation in thenucleus were visualized using a Bio-Rad MRC1024 confocal laser scanningmicroscope and LaserSharp2000 acquisition software.

FIG. 10 shows the relative cell count for the presence of ribonuclearfoci in the nucleus of DM500 myoblasts transfected with PS58 or mockcontrol from the experiment depicted in FIG. 9.

FIG. 11 shows the RT-PCR analysis of hDMPK mRNA in muscle of DM500 micetreated with PS58 or mock control. Shortly, PS58 (2 nmol) was injectedin the GPS complex of one-year-old DM500 mice and this procedure wasrepeated after 24 h. After 15 days, M. plantaris and M. gastrocnemiuswere isolated and RT-PCR was performed on total RNA for hDMPK and mouseactin. The intensity of the hDMPK signal was normalized to the actinsignal and the values expressed relative to mock control.

FIG. 12 shows a Northern blot analysis of DM500 myotubes treated withdifferent oligonucleotides (200 nM) or mock control. PS58, PS146 andPS147 carried a full 2′O-methyl phosphorothiate backbone, but differedin length, (CAG)7, (CUG)10 and (CUG)5, respectively. PS142 has acomplete phosphorothiate DNA backbone with a (CAG)7 sequence. hDMPK andmDMPK signals were normalized to mouse GAPDH and expressed as percentageto mock control. Quantification is shown in the lower panel.

EXAMPLES Example 1

Immortomyoblast cell lines were derived from DM500 or CTG110 mice usingstandard techniques known to the skilled person. DM500 mice were derivedfrom mice obtained from de Gourdon group in Paris. CTG110 mice aredescribed below and present at the group of Wiering a and Wansink inNijmegen. Immortomyoblast cell lines DM500 or CTG 110 with variable(CTG)n repeat length in the DMPK gene were grown subconfluent andmaintained in a 5% CO₂ atmosphere at 33° C. on 0.1% gelatin coateddishes. Myoblast cells were grown subconfluent in DMEM supplemented with20% FCS, 50 μg/ml gentamycin and 20 units of γ-interferon/ml. Myotubeformation was induced by growing myoblast cells on Matrigel (BDBiosciences) coated dishes and placing a confluent myoblast culture at37° C. and in DMEM supplemented with 5% horse serum and 50 μg/mlgentamycin. After five days on this low serum media contracting myotubesarose in culture and were transfected with the desired oligonucleotides.For transfection NaCl (500 mM, filter sterile), oligonucleotide andtransfection reagens PEI (ExGen 500, Fermentas) were added in thisspecific order and directly mixed. The oligonucleotide transfectionsolution contained a ratio of 5 μl ExGen500 per ug oligonucleotide whichis according to the instructions (ExGen 500, Fermentas). After 15minutes of incubation at room temperature the oligonucleotidetransfection solution was added to the low serum medium with thecultured myotubes and gently mixed. The final oligonucleotideconcentration was 200 nM. Mock control treatment is carried out withtransfection solution without an oligonucleotide. After four hours ofincubation at 37° C., fresh medium was added to the culture (resultingin a dilution of approximately 2.3×) and incubation was extendedovernight at 37° C. The next day the medium containing theoligonucleotide was removed and fresh low serum medium was added to themyotubes which were kept in culture at 37° C. for another day. Fourtyeight hours after the addition of oligonucleotide to the myotube culture(which is seven days after switching to low serum conditions to inducedmyotube formation), RNA was isolated with the “Total RNA mini kit”(Bio-Rad) and prepared for Northern blot and RT-PCR analysis. TheNorthern blot was hybridized with a radioactive human DMPK (hDMPK) probeand a mouse GAPDH probe. The probe used for DMPK is a human DMPK cDNAconsisting of the DMPK open reading frame with full 3′ UTR and 11 CTGs.

The human and mouse DMPK signal were quantified by phosphoimageranalysis and normalized to the GAPDH signal. Primers that were used forthe RT-PCR for hDMPK mRNA were situated in the 3′ untranslated part withthe sequence 5′-GGGGGATCACAGACCATT-3′ and 5′-TCAATGCATCCAAAACGTGGA-3′and for murine actin the primers were as followed: Actin sense5′-GCTAYGAGCTGCCTGACGG-3′ and Actin antisense 5′-GAGGCCAGGATGGAGCC-3′.PCR products were run on an agarose gel and the signal was quantifiedusing Labworks 4.0 (UVP BioImaging systems, Cambridge, United Kingdom).The intensity of each band was normalized to the intensity of thecorresponding actin band and expressed relative to mock control.

Thirteen different oligonucleotides were tested (for an overview seeTable 1) as described above on the immortomyoblast DM500 cell linecontaining transgenic human DMPK gene with (CTG)n repeat length ofapproximately 500 and a normal mouse DMPK gene without (CTG) repeat.FIG. 1 shows the Northern blot of the isolated RNA from theoligonucleotide transfected myotubes visualized with the hDMPK probe anda GAPDH probe for loading control. Quantification of the human DMPK(with CTG repeat) and murine DMPK (without CTG repeat) signal on theNortherm blot is shown in FIG. 2. The signal was normalized to murineGAPDH and expressed relative to mock control.

Table 2 indicates the level of hDMPK mRNA reduction that is caused by aspecific oligonucleotide. The minus (−) stands for no reduction and thenumber of positive signs (+) stands for the relative level of hDMPK mRNAbreak-down. Clearly, oligonucleotide PS58, specifically targeted to therepeat sequence, is much more potent in reducing or altering hDMPKtranscripts than the other oligonucleotides complementary to uniquesequences in the hDMPK transcripts.

FIG. 3 shows the effect of PS58 in murine immortomyotubes derived fromCTG110 mice, a knock-in mouse containing a DMPK gene with the 3′ part ofthe human DMPK gene including a (CTG) repeat of approximately 110.Northern blot analysis showed that the DMPK transcript containing the(CTG)110 repeat was reduced by the treatment with oligonucleotide PS58but not after mock treatment.

Example 2 FIG. 4

The DM500 immortomyoblast cell line carrying a human DMPK gene with anapproximate (CTG)500 repeat expansion was cultured, prepared andtransfected as described above (see example 1). In this example, thetransfection was carried out with PS58 at different concentrations.Eighty four hours after start of treatment, the myotubes were harvestedand Northern blot analysis was performed on isolated RNA as describedabove (see example 1).

FIG. 4 shows the quantification of the hDMPK mRNA signal preformed byphosphoimager analysis and normalized to the GAPDH signal at differentconcentrations. Under these conditions, a half maximal effect wasobserved at around 1 nM.

Example 3 FIGS. 5 and 6

The DM500 immortomyoblast cell line carrying a human DMPK gene with anapproximate (CTG)500 repeat expansion was cultured, prepared andtransfected as described above (see example 1). However, in this examplethe transfection with 200 nM PS58 was carried out at different timepoints. Usually DM500 myotubes were harvested seven days after switchingto low serum conditions to induce myotube formation. The standardprocedure (as in example 1 and 2) was to start treatment (transfection)48 h (two days) before harvesting. Now, treatment with PS58 was started2 h-48 h (FIG. 5) or 2 d-8 d (FIG. 6) before harvesting. Northern blotanalysis and quantification was performed as before.

FIG. 5 shows that expanded hDMPK mRNA in DM500 myotubes was decreasedrapidly within 2 h of treatment with oligonucleotide PS58 compared tomock control treatment.

FIG. 6 shows a persistent decrease in expanded hDMPK mRNA in DM500myotubes for at least 8 days. Please note that in the case of the 8 dexperiment, cells were transfected in the myoblast stage (approximately60% confluent, 33° C., high serum) and that they have received freshmedium on various occasions until harvesting (including a change to lowserum at 37° C., two days after transfection). Example 2 and 3 areindicative of a highly efficient inhibitory intervention by anoligonucleotide directed solely to the repeat expansion. The magnitudeof this effect might be influenced by the relative low levels of hDMPKexpression in these model cell systems, which normally is also seen inhumans.

Example 4 FIG. 7

In this example, fibroblasts obtained from a human patient withcongenital myotonic dystrophy type 1 (cDM1) were used. These patientcells carry one disease causing DMPK allele with a triplet repeatexpansion length of 1500 and one normal DMPK allele with a repeat lengthof 11. The size of the (CTG)n expansion on both alleles was confirmedwith PCR and Southern blotting.

The fibroblasts were grown sub confluent and maintained in a 5% CO₂atmosphere at 37° C. on 0.1% gelatin coated dishes. Fibroblasts weregrown subconfluent in DMEM supplemented with 10% FCS and 50 μg/mlgentamycin. Myotube formation was induced by growing fibroblasts cellson Matrigel (BD Biosciences) coated dishes and infecting the cells at75% confluency with MyoD-expressing adenovirus (Ad5Fib50MyoD, Crucell,Leiden) (MOI=100) in DMEM supplemented with 2% HS and 50 μg/mlgentamycin for 2 hours. After the incubation period MyoD adenovirus wasremoved and DMEM supplemented with 10% FCS and 50 μg/ml gentamycin wasadded. The cells were maintained hi this medium in a 5% CO₂ atmosphereat 37° C. until 100% confluency. At this point cells were placed in DMEMsupplemented with 2% FCS and 50 μg/ml gentamycin. After five days onthis low serum media cells were transfected with PS58 following theprocedure according to the instructions (ExGen 500, Fermentas) and asdescribed above. The final oligonucleotide concentration was 200 nM and20 nM. Fourty eight hours after start of the treatment (which is sevendays after switching to low serum conditions), RNA was isolated with the“Total RNA mini kit” (Bio-Rad) and prepared for Northern blot. TheNorthern blot was hybridized with a radioactive human DMPK (hDMPK) andmouse GAPDH mRNA probe. The human DMPK signals were quantified byphosphoimager analysis and normalized to the GAPDH signal and expressedrelative to mock control.

FIG. 7 shows the Northern blot analysis of the MyoD-transformedmyoblasts treated with oligonucleotide PS58 (20 and 200 nM). The resultsdemonstrate an effective complete inhibition of the disease-causinghDMPK (CUG)1500 RNA transcript, while the smaller normal hDMPK (CUG)11RNA transcript is only moderately affected at the two concentrations.Thus, oligonucleotides directed to the repeat region exhibit selectivitytowards the larger repeat size (or disease causing expansion).

Example 5 FIG. 8

In this example, the DM500 immortomyoblast cell line carrying a humanDMPK gene with an approximate (CTG)500 repeat expansion was cultured,transfected and analysed as described before in example 1. The DM500myotubes were treated 48 h before harvesting with 200 nM ofoligonucleotide PS58, specific to the (CUG) repeat sequence only,oligonucleotide PS113, specific to a sequence in exon 1, or mockcontrol. RT-PCR analysis was performed on hDMPK mRNA expressed in thismurine cell line (for primers see example 1) and on three other genetranscripts with a naturally occurring (CUG) repeat in mice, Ptbp1 witha (CUG)6, Syndecan3 with a (CUG)6 and Taxilinbeta with a (CUG)9.

The PCR primers used were for Ptbp1: 5′-TCTGTCCCTAATGTCCATGG-3′ and5′-GCCATCTGCACAAGTGCGT-3′; for Syndecan3: 5′-GCTGTTGCTGCCACCGCT-3′ and5′-GGCGCCTCGGGAGTGCTA-3′; and for Taxilinbeta: 5′-CTCAGCCCTGCTGCCTGT-3′and 5′-CAGACCCATACGTGCTTATG-3′. The PCR products were run on an agarosegel and signals were quantified using the Labworks 4.0 program (UVPBioImaging systems, Cambridge, United Kingdom). The intensity of eachsignal was normalized to the corresponding actin signal and expressedrelative to mock control.

FIG. 8 shows the RT-PCR results with a maximal inhibition of hDMPK mRNAexpression by PS58. The other gene transcripts carrying a naturallyoccurring small (CUG) repeat were not or only marginally affected by theoligonucleotide PS58, specific to the (CUG) repeat, compared tooligonucleotide PS113, which has no complementary sequence to these genetranscripts.

This example confirms the selectivity of an oligonucleotide, directedsolely to the repeat region, towards the long repeat size (or diseasecausing expansion) compared to naturally occurring shorter repeat sizes.

Example 6 FIGS. 9 and 10

In this example, the DM500 immortomyoblast cell line carrying a humanDMPK gene with an approximate (CTG)500 repeat expansion was cultured andtransfected with PS58 (200 nM). Here, FISH analysis was carried out onthe cells. Fourty eight hours after the start of the treatment, thecells were fixed with 4% formaldehyde, 5 mM MgCl₂ and 1×PBS for 30minutes. Hybridization with fluorescently labeled oligonucleotideCy3-(CAG)10-Cy3 was performed overnight at 37° C. in a humid chamber.After hybridization the material was washed and mounted in mowiol andallowed to dry overnight. Nuclear inclusions (ribonuclear foci) werevisualized using a Bio-Rad MRC1024 confocal laser scanning microscopeand LaserSharp2000 acquisition software. In total 50 cells were countedand scored for the presence of inclusions in the nuclei of these cells.

Literature indicates that DMPK mRNA containing a (CUG) expanded repeataccumulates and aggregates in the nucleus to form ribonuclear foci withregulatory nuclear proteins and transcription factors. Therefore, normalnuclear gene processing and cell function gets impaired.

FIG. 9 shows a mock treated cell containing ribonuclear inclusions inthe nucleus, while these are no longer present in the cell nucleus aftertreatment with PS58. FIG. 10 shows that the percentage of nucleicontaining ribonuclear foci seen under control conditions in DM500myotubes is strongly decreased by the treatment with PS58. This resultdemonstrates that inhibition of hDMPK mRNA expression also inhibits thedisease related triplet repeat (CUG) rich inclusions.

Example 7 FIG. 11

Here, the effect of PS58 was evaluated in vivo in DM500 mice containinghDMPK with a (CTG)n expansion of approximately 500 triplets. The DM500mice were derived by somatic expansion from the DM300 mouse (e.g. seeGomes-Pereira M et al (2007) PLoS Genet. 2007 3(4): e52). A (CTG)triplet repeat expansion of approximately 500 was confirmed by southernblot and PCR analysis.

In short, PS58 was mixed with transfection agent ExGen 500 (Fermentas)according to the accompanying instructions for in vivo use. PS58 (2nmol, in the transfection solution with Exgen 500) was injected (40 μl)in the GPS complex of one-year-old DM500 mice and this procedure wasrepeated after 24 h. As a control, DM500 mice were treated similarlywith the transfection solution without PS58. After 15 days, the micewere sacrificed, muscles were isolated and total RNA was isolated fromthe tissues (using Trizol, Invitrogen). RT-PCR analysis was performed todetect hDMPK mRNA in the muscle similar as described above. Theintensity of each band was performed using the Labworks 4.0 program (UVPBioImaging systems, Cambridge, United Kingdom) and normalized to theintensity of the corresponding actin band. Primer location is indicatedin the figure.

FIG. 11 shows that in vivo treatment of DM500 mice with PS58 stronglyreduced the presence of hDMPK mRNA containing a (CUG)n repeat expansioncompared to mock treatment in the M. plantaris and M. gastrocnemius.

Example 8 FIG. 12

In this example, different oligonucleotides (in length and backbonechemistry) but all with a sequence directed solely to the (CTG)n repeatexpansion were compared. DM500 myotubes were cultured, transfected andanalysed as described above in example 1. Northern blots were quantifiedby phosphoimager analysis and DMPK signals were normalized to GAPDH.

Here, the DM500 myotubes were treated with the followingoligonucleotides (200 nM), all with a complete phosphorothioate backbone(see Table 3).

FIG. 12 shows that treatment of the DM500 myotubes results in a completereduction of (CUG)n expanded hDMPK mRNA for all oligonucleotides tested.Under the present conditions, the maximal effect obtainable isindependent of oligonucleotide length, backbone modification orpotential mechanism of inhibition by the employed single strandedoligonucleotides.

Example 9

Fibroblasts (GM 00305) from a male patient with Huntington's Diseasewere obtained from Coriell Cell Repository (Camden, N.J., US) andcultured according to the accompanying instructions and standardtechniques known to the skilled person in the art. Huntington patientscarry one healthy and one disease-causing allele of the Huntington generesulting in the expression of both mRNAs with respectively a normalnumber and an expanded number of (CAG) repeats, respectively.

The fibroblasts were transfected with a 21-mer 2′O-methylphosphorothioate RNA antisense oligonucleotide PS57 with a (CUG)7sequence, complementary to the (CAG) triplet repeat in Huntington mRNA.Transfection occurred at 100 or 200 nM in the presence of PEI asindicated by the manufacturer. Twenty four hours after transfection thecells were harvested and total RNA was isolated and analysed by RT-PCR.The Huntington transcript was determined using primers in downstreamexon 64 (5′ GAAAG TCAGT CCGGG TAGAA CTTC 3′ and 5′ CAGAT ACCCG CTCCATAGCA A 3′). This method detects both types of Huntington mRNAs, thenormal and mutant transcript with the additional (CAG) expansion. GAPDHmRNA (housekeeping gene) was also determined. The signals werequantified and the total amount of Huntington mRNA was normalised to theamount of GAPDH mRNA in the same sample. The results are expressedrelative to a control treated (without oligonucleotide) sample fromfibroblasts (which was to 100%).

In the samples from fibroblasts transfected with either 100 or 200 μM ofPS57, significantly lower levels of total Huntington mRNA levels wereobserved of approximately 53% and 66% compared to the levels incontrol-treated cells, respectively.

Thus, PS57, an oligonucleotide directed only to the (CAG) repeat,induces a decrease in Huntington mRNA levels and these results areconsistent with a selective inhibition of mutant over normal HuntingtonmRNA.

TABLE 1 Overview oligonucleotides tested Oligo name ModificationSequence Position PS40 2′OMe RNA phosphorothioate/FAMGAGGGGCGUCCAGGGAUCCG intron 14-exon 15 PS41 2′OMe RNA phosphorothioateGCGUCCAGGGAUCCGGACCG intron 14-exon 15 PS42 2′OMe RNA phosphorothioateCAGGGAUCCGGACCGGAUAG intron 14-exon 15 PS56 DNA CAGCAGCAGCAGCAGCAGCAGrepeat in exon 15 PS58 2′OMe RNA phosphorothioate/FAMCAGCAGCAGCAGCAGCAGCAG repeat in exon 15 PS59 2′OMe RNA phosphorothioateUGAGUUGGCCGGCGUGGGCC ESE exon 15 PS60 2′OMe RNA phosphorothioateUUCUAGGGUUCAGGGAGCGCGG ESE exon 15 PS61 2′OMe RNA phosphorothioateACUGGAGCUGGGCGGAGACCC ESE exon 15 PS62 2′OMe RNA phosphorothioateCUCCCCGGCCGCUAGGGGGC ESE exon 15 PS113 DNA phosphothioroateGAGCCGCCTCAGCCGCACCTC Exon 1 PS114 DNA phosphothioroateGAAGTCGGCCACGTACTTGTC Exon 1 P8115 DNA phosphothioroateGGAGTCGAAGACAGTTCTAGG Exon 15 PS116 DNA phosphothioroateGGTACACAGGACTGGAGCTGG Exon 15

TABLE 2 Reduction of hDMPK mRNA after oligo transfection: OligoReduction hDMPK mRNA SEQ ID No.'s PS40 + 1 PS41 − 2 PS42 − 3 PS59 − 4PS60 − 5 PS61 +/− 6 PS62 − 7 PS58 ++++ 8 PS56 − 9 PS113 − 10 PS114 − 11PS115 +/− 12 PS116 + 13 (−) indicates no reduction, (+) indicates levelof reduction in hDMPK mRNA.

TABLE 3 Oligonucleotides used in example 9 RNAse H breakdown # Length(CAG)n Substitution ribose possible PS58 21-mer n = 7 2′O-Methyl NoPS146 30-mer n = 10 2′O-Methyl No PS147 15-mer n = 5 2′O-Methyl No PS14221-mer n = 7 Deoxyribose (DNA) Yes *all oligonucleotides full lengthphosphorothioate and substitution

1. A method for preventing or treating a genetic disorder in a subject,comprising administering to a subject with a genetic disorder that isassociated with human cis-element repeat instability a single strandedoligonucleotide comprising or consisting of a sequence that iscomplementary only to a repetitive element sequence in a gene transcriptof a gene with said repeat instability.
 2. The method according to claim1 wherein the repetitive element is present in a coding sequence of thegene transcript.
 3. The method according to claim 1 wherein therepetitive element is present in a non-coding sequence of the genetranscript.
 4. The method according to claim 1 wherein the sequence ofthe repetitive element is selected from the group consisting of CAG;GCG; CUG; CGG; CCG; GAA; GCC; and CCUG.
 5. The method according to claim2, wherein the oligonucleotide comprises or consists of a sequence thatis complementary to a CAG repeat and wherein the disorder isHuntington's disease, spino-cerebellar ataxias, Haw River syndrome,X-linked spinal and bulbar muscular atrophy ordentatorubral-pallidoluysian atrophy.
 6. The method according to claim2, wherein the oligonucleotide comprises or consists of a sequence thatis complementary to a GCG repeat and wherein the disorder is infantilespasm syndrome, deidocranial dysplasia, blepharophimosis,hand-foot-genital disease, synpolydactyl, oculopharyngeal musculardystrophy or holoprosencephaly.
 7. The method according to claim 3,wherein the oligonucleotide comprises or consists of a sequence that iscomplementary to a CUG repeat and wherein the disorder is myotonicdystrophy type 1, spino-cerebellar ataxia 8 or Huntington's disease-like2.
 8. The method according to claim 3 wherein the oligonucleotidecomprises or consists of a sequence that is complementary to a CCUGrepeat and wherein the disorder is myotonic dystrophy type
 2. 9. Themethod according to claim 3, wherein the oligonucleotide comprises orconsists of a sequence that is complementary to a CGG repeat and whereinthe disorder is fragile X syndrome.
 10. The method according to claim 3,wherein the oligonucleotide comprises or consists of a sequence that iscomplementary to a GAA repeat and wherein the disorder is Friedreich'sataxia.
 11. The method according to claim 1 wherein the oligonucleotidehas a length of about 10 to about 50 nucleotides.
 12. The methodaccording to claim 1 wherein the single stranded oligonucleotidecomprises ribonucleotides, deoxyribonucleotides, nucleotides of a lockednucleic acid (LNA), nucleotides of a peptide nucleic acid (PNA),morpholino phosphorodiamidates, nucleotides of an ethylene-bridgednucleic acid or a mixture thereof.
 13. The method according to claim 12,wherein the oligonucleotide comprises 2′-O-substituted RNAphosphorothioate nucleotides.
 14. The method according to claim 1wherein the administered oligonucleotide is in the form of anexpressible nucleic acid vector.
 15. The method according to claim 1,wherein the oligonucleotide is in a pharmaceutical composition whichfurther comprises an excipient and/or targeting ligand that delivers theoligonucleotide to cells and/or enhances intracellular delivery of theoligonucleotide.
 16. A single stranded oligonucleotide comprising orconsisting of a sequence of about 10 to about 50 nucleotides that iscomplementary to a repetitive sequence of a tri- or tetranucleotideselected from the group consisting of (a) CAG; (b) GCG; (c) CUG; (c)CGG; (d) GAA; (e) GCC; (f) CCUG.
 17. The oligonucleotide according toclaim 16, comprising 2′-O-substituted phosphorothioate ribonucleotides,phosphorothioate deoxyribonucleotides, LNA nucleotides, morpholinonucleotides, and/or combinations thereof.
 18. The oligonucleotideaccording to claim 26 wherein the label is a radioactive label or afluorescent label.
 19. A pharmaceutical composition comprising anoligonucleotide according to claim 16, and a pharmaceutically acceptableexcipient.
 20. The pharmaceutical composition of claim 19, furthercomprising a targeting ligand that delivers the oligonucleotide to acell and/or enhances intracellular delivery of the oligonucleotide. 21.A nucleic acid vector, that expresses the oligonucleotide according toclaim 16 in human cells.
 22. A method for reducing the number ofrepeat-containing gene transcripts in a cell comprising providing tosaid cell the oligonucleotide according to claim
 16. 23. (canceled) 24.The method according to claim 11 wherein the oligonucleotide has alength of about 12 to about 30 nucleotides.
 25. The method of claim 12wherein the single stranded oligonucleotide has aphosphorothioate-containing backbone.
 26. The oligonucleotide of claim16 that further comprises a detectable label.
 27. A method for detectingthe presence of nucleic acid repetitive elements in cells, comprising:(a) contacting the cells or a lysate or extract thereof with theoligonucleotide according to claim 18, under conditions wherein saidoligonucleotide hybridizes with cellular DNA and/or RNA; (b) detectinghybridization of said oligonucleotide, wherein the hybridization of saidoligonucleotide is indicative of the presence of said repeat elements insaid cells.
 28. A method for diagnosing a genetic disorder associatedwith human cis-element repeat instability in a subject, comprisingperforming the method of claim 27 on cells, or obtained from a subjectwith, suspected of having, or at risk for said disorder, or on a lysateor extract of said cells, wherein detection of the presence of saidrepeat elements in said cells, lysate or extract is diagnostic of saidgenetic disorder.